The role of MLO in powdery mildew susceptibility depends on a combination of functional specialization and subcellular localization

Abstract

Obligate biotrophic powdery mildew (PM) fungi strictly require living hosts to survive. To search for host factors or processes essential for PM pathogenesis, we conducted a tailored forward genetic screen with the immuno-compromised eds1-2/pad4-1/sid2-2 (eps) triple Arabidopsis mutant. This led to the identification of five allelic disruptive mutations in Mildew Locus O 2 (MLO2) that are responsible for the compromised-immunity-yet-poor infection (cipi) mutant phenotype upon challenge with an adapted PM isolate. Moreover, the eds1/pad4/sid2/mlo2/mlo6/mlo12 (eps3m) sextuple and the eds1/pad4/sid2/pen1/pen2/pen3/mlo2/mlo6/mlo12 (eps3p3m) nonuple mutants displayed near-complete immunity to adapted and non-adapted PM fungi without signs of defense activation, further strengthening the inference that these three clade V MLOs in Arabidopsis may be bona fide host susceptibility factors of PM fungi. Confocal imaging revealed focal accumulation of MLO2-GFP in the peri-penetration peg membranous space, which occurs before and may be required for haustorium differentiation. Ectopic leaf expression analyses of eight other MLOs belonging to different clades showed that only MLO7 can complement the loss of MLO2, MLO6, and MLO12. Results from domain-swapping analyses between MLO1 and MLO2 suggest a bipartite functional configuration for MLO2: its cytoplasmic C-terminus determines where and when MLO2 functions, while its N-terminal seven transmembrane domain-containing region executes the cellular function that is critical for PM pathogenesis. Genetic studies further demonstrated that, unlike MLO7 in synergids, focal accumulation of MLO2 does not depend on FERONIA (FER) and its five paralogs. Together, these findings define clade V MLOs as host factors co-opted by obligate biotrophic PM fungi for successful host colonization.